Recombinant adenoviral vectors and applications thereof

ABSTRACT

The invention relates to novel recombinant adenoviruses which can be obtained from a replicating adenovirus by deleting all or part of the region of the genome of said replicating adenovirus corresponding to that located in the genome of canine adenovirus type 2 (GenBank J04368) between positions  311  and  499 , the aforementioned deletion comprising all or part of the region of the genome of the original replicating adenovirus corresponding to that located between positions  311  and  401  in the genome of canine adenovirus type 2. The invention also relates to the uses of said adenoviruses, particularly for therapeutic purposes.

The present invention relates to novel recombinant adenoviruses and themethod for preparing them, as well as to their uses as expression andgene transfer vectors for vaccinatory purposes or for therapeuticpurposes such as the treatment of cancer.

Adenoviruses are naked viruses which possess a linear double-strandedDNA genome of about 30-40 kbp in size, flanked by short inverted repeatsequences (ITRs).

The genome of the adenovirus is organized in early transcription units(E1 to E4) and a late unit (MLTU) which is composed of five transcriptfamilies (L1 to L5) whose expression is separated by the initiation ofthe replication of the viral DNA.

The early phase begins, two hours after infection, with thetranscription and sequential expression of the regions E1A then E4,almost simultaneously E3 and E1B, then E2A and finally E2B. Theimmediate early region E1 A encodes transactivators of other early genesof the adenovirus (E1 B, E2, E3 and E4) as well as of cellular genes.Replication of the viral DNA begins eight hours after infection. Thelate phase, which commences twelve hours after infection, ischaracterized by abolition of the synthesis of the cellular proteins infavor of the late viral proteins, which enter into the structuralcomposition of the adenoviral particle and participate in the assemblyof the virion and its release while modifying the structural integrityof the infected cell.

The adenoviruses are particularly attractive for developing viralvectors due to their characteristics and the amount of knowledge whichis available with regard to their genetic organization and theirbiology.

Different construction strategies have been considered depending onwhether the aim is to obtain a replicating virus, which is able tomultiply in the host (human or animal), or a nonreplicating virus whichis unable to multiply in the host.

Constructing a nonreplicating vector involves deleting a region which isessential for viral replication. The resulting viruses, which are unableto replicate and consequently to produce infectious particles in cellswhich are permissible for infection by the corresponding wild-typevirus, are produced in modified cell lines which are able to supply theproducts of the deleted genes in trans.

A strategy which is commonly used consists in inserting a heterologousgene into the left-hand part of the genome between the left ITR and theE1 region in place of the promoter and of the coding region of the E1Agene (partial E1 deletion) and, where appropriate, of the E1B gene(total deletion of the E1 region). The viruses from which E1A has beendeleted are unable to replicate in cells which do not complement the E1Afunctions. However, they are able to express substantial quantities ofexogenous protein in the infected cells.

A large number of human adenoviral vectors (Ad2 and Ad5) in which the E1region and, where appropriate, the E3 region are deleted have beenconstructed, mainly for the purpose of human gene therapy. Mutations (E2region) or additional deletions (E2 or E4 region) have been introducedfor the purpose of improving these vectors.

Nonreplicating canine adenoviral vectors in which all the E1 region hasbeen deleted have also been developed for human gene therapyapplications [Klonjkowski et al., Human Gene Therapy, 8, 2103-2115,1997, deletion of positions 411 to 2898 of Cav2; application WO 95/14101and U.S. Pat. No. 5837531 in the name of Rhone Poulenc Rorer; Kremer etal., J. Virol., 74, 505-512, 2000, deletion of positions 412 to 2497 ofCav2).

These nonreplicating vectors have demonstrated good efficiency intransferring genes into a large number of tissues. However, they sufferfrom a certain number of disadvantages in particular for transferringgenes into actively dividing cells such as tumor cells. In these cells,rapid abolition of the expression of the transferred gene is observed,with this abolition being linked to the loss of the extrachromosomalvector during the course of successive divisions.

Constructing a replicating vector involves the necessity of noteliminating any sequence in the viral genome which is essential for itsreplication and for the production of infectious viral particles in thehost (productive viral cycle). Only a small number of heterologoussequence insertion sites which satisfy these requirements are at presentknown in adenoviruses.

Replicating vectors have been obtained by inserting heterologous genesinto nonessential regions such as the E3 region and the right-hand partof the genome between the right ITR and the transcriptional regulationsequences of the E4 promoter. Replicating vectors have also beenobtained by inserting a heterologous gene into the left-hand part of thegenome between the left ITR and the E1 region provided that functionalE1 genes are preserved. More precisely, insertion of a heterologoussequence between positions 455 and 917 of the human adenovirus (Ad5),which inactivates the E1A gene by deleting the promoter and a part ofthe coding region of E1A, is compensated by inserting a copy of thisgene into the vector in an ectopic position (E1 oit et al., J. Gen.Virol., 76, 1583-1589, 1995).

Replicating vectors have been constructed in this way from human (E1 oitet. al., see above), bovine (Mittal et al., J. Gen. Virol., 76, 93-102,1995), ovine (Xu et al., Virology, 230, 62-71, 1997), avian (Michou etal., J. Virol., 73, 1399-1410, 1999; Sheppard et al., Arch. Virol., 143,915-930, 1998), canine (Cav2; international application WO 98/00166 andU.S. Pat. No. 6090393, in the name of Rhone Merieux; internationalapplication WO 91/11525 and U.S. Pat. No. 5616326 in the name of GlasgowUniversity, Morrison et al., Virology, 293, 26-30, 2002) and porcine(Reddy et al., J. Gen. Virol., 80, 563-570, 1999; Tuboly et al., J. Gen.Virol., 82, 183-190, 2001) adeno-viruses.

These replicating adenoviruses have been mainly developed forvaccinatory applications. In a general manner, they have demonstrated ahigh level of efficacy in connection with inducing immune responses,both as far as the antibody response and the CTL response are concerned(for a review, see E1 oit, Virologie [Virology], 2, 109-120, 1998 andKlonjkowski et al., in “Adenoviruses: basic biology to gene therapy”,pp. 163-173, P. Seth, Ed., R.G. Landes Company, Austin Tex., USA).

However, these replicating vectors suffer from some drawbacks:

-   -   they pose problems of biosafety linked to the risk of these        replicating viruses spreading,    -   the substantial quantity of the viral particles which is        produced by the infected cells leads to a powerful immune        response being induced against the vector and limits the        efficacy of repeat injections,    -   neutralization by the maternal antibodies of the vaccinatory        antigen which is released from cells which have been destroyed        by the infection decreases the efficacy of these replicating        vectors in the young animal.

It is apparent that no recombinant adenovirus which makes it possible toefficiently transduce cells, in particular dividing cells such as tumorcells, without involving risks of dissemination into the environment iscurrently available.

In choosing type 2 canine adenovirus (Cav2) as an experimental model,the inventors sought to determine whether it was possible to identifynew insertion sites which made it possible to obtain replicatingrecombinant adenoviruses.

Thus, they observed that deleting a small proportion of the beginning ofthe region located between the end of the left ITR and the beginning ofthe sequence encoding E1A did not affect the ability of the adenovirusesto replicate their genome and to multiply in a permissive host, the siteof this deletion can therefore constitute a novel site for insertingheterologous genes.

In addition, the inventors observed that, surprisingly, other deletionsin the same region made it possible to obtain adenoviruses which wereable to replicate their genome in a permissive host but which wereunable to multiply. These adenoviruses will be designated“pseudoreplicating adenoviruses” below.

In that which follows, the positions of the different regions of theadenoviral genome are defined by reference to the positions of thecorresponding regions (that is to say, containing elements having asimilar function) of the genome of the type 2 canine adenovirus in theGenBank J04368 sequence.

Thus, the region located between the end of the left ITR and thebeginning of the sequence encoding E1A corresponds to that locatedbetween position 311 and position 499 in the GenBank J04368 genomicsequence of type 2 canine adenovirus.

The present invention relates to a recombinant adenovirus which can beobtained from a replicating adenovirus by deleting all or part of theregion of the genome of said replicating adenovirus which corresponds tothat located between positions 311 and 499 in the genome of type 2canine adenovirus (GenBank J04368), with said deletion comprising all orpart of the region of the genome of the original replicating adenoviruscorresponding to that located between positions 311 and 401 in thegenome of type 2 canine adenovirus.

According to a first embodiment of a recombinant adenovirus according tothe invention, the deleted portion consists of all or part of the regionof the genome of the original replicating adenovirus which correspondsto that located between positions 311 and 319 in the genome of type 2canine adenovirus; this deletion makes it possible to obtain areplicating recombinant adenovirus which is able to multiply in a hostwhich is permissive to infection with an original wild-type adenovirus(productive viral cycle).

According to a second embodiment of a recombinant adenovirus accordingto the invention, the deleted portion comprises all or part of theregion of the genome of the original replicating adenovirus whichcorresponds to that located between positions 318 and 401 in the genomeof type 2 canine adenovirus; this deletion advantageously makes itpossible to obtain pseudoreplicating adenoviruses, that is to sayadenoviruses which are able to replicate but unable to produceinfectious viral particles and therefore unable to multiply (abortivecycle) in a host which is permissive to infection with the originalwild-type adenovirus.

Obtaining pseudoreplicating adenoviruses according to the inventioninvolves, in particular, eliminating all or part of the putativeencapsidation signals of the 5′-TTTA/G-340 A_(X), A_(XI), and A_(XII)type (respectively located in positions 341-344, 377-380 and 388-391 inthe GenBank J04368 Cav2 sequence).

The portion which is deleted in these pseudoreplicating adenoviruses canadditionally comprise:

-   -   all or part of the region of the genome of the original        replicating adenovirus corresponding to that located between        positions 311 and 319 in the genome of type 2 canine adenovirus;        and/or    -   all or part of the region of the genome of the original        replicating adenovirus corresponding to that located between        positions 400 and 439 in the genome of type 2 canine adenovirus;        this deletion eliminates, in particular, the TATA box of the E1A        promoter (located in position 409 in the GenBank J04368 Cav2        sequence); and/or    -   all or part of the region of the genome of the original        replicating adenovirus corresponding to that located between        positions 438 and 499 in the genome of type 2 canine adenovirus;        this deletion eliminates, in particular, the E1A transcription        initiation site (located in position 439 in the GenBank J04368        Cav2 sequence).

In all these cases, the (replicating or pseudo-replicating) recombinantadenoviruses according to the invention retain the left ITR sequenceswhich are essential for replication and for activating transcription (4repeated GGTCA motifs located between positions 62 and 99 in the Cav2genome) as well as the 5′TTGN₈CG-340 type A_(I) and 5′-TTTA/G-3′ typeA_(II) to A_(IX) encapsidation signals (respectively located atpositions 197-200, 206-209, 213-216, 226-232, 239-242, 250-253, 258-261,272-275 and 306-309 in the Cav2 genome). They also retain all of the E1Acoding sequence as well as regions of the E1 gene located downstreamthereof (E1A polyadenylation signal and E1B region).

According to a preferred embodiment of a recombinant adenovirusaccording to the invention, it additionally comprises at least oneheterologous sequence of interest inserted in its genome.

In order to construct a recombinant adenovirus in accordance with thisembodiment, said heterologous sequence will, in the case of areplicating adenovirus, be inserted into the region of the genomecorresponding to that located between positions 311 and 319 in thegenome of type 2 canine adenovirus.

In the case of a pseudoreplicating adenovirus, said heterologoussequence can also be inserted into this region or at any other site inthe region of the genome corresponding to that located between positions311 and 499 in the genome of type 2 canine adenovirus. The insertioninto this region can be effected in place of the deleted portion or inthe vicinity thereof.

A heterologous sequence can also be inserted into any one of the siteswhich are normally used for this purpose for constructing replicatingadenoviruses. The insertion can, for example, be effected in the E3region or in the region located between the E4 region and the right ITR,as described in U.S. Pat. No. 6090393, or in the 3′ portion of the rightITR, as described in U.S. Pat. No. 5616326.

A “heterologous sequence” is understood as meaning any sequence otherthan that contained between positions 311 and 499 in the genome of saidwild-type adenovirus.

The following may be mentioned as nonlimiting examples:

-   -   - genes encoding a vaccinal antigen, for example the gag or env        genes of the feline immunodeficiency virus (FIV), the S, M or N        protein of the feline coronavirus, a canine or feline parvovirus        capsid protein, the G glycoprotein of the rabies virus or the        Leptospira sp. Hap-1 protein, etc.    -   corrective genes which can be used in gene therapy, for example        that for erythropoietin (Epo), for vascular endothelium growth        factor (VEGF), for neurotrophin 3 (NT-3) or for atrial        natriuretic factor (ANF), etc.;    -   genes which can be used for treating cancer, for example that        for IL-2, that for IFNγ, etc.

Recombinant adenoviruses according to the invention can, in particular,be derived from mammalian adenoviruses and, in particular, canineadenoviruses, in particular type 2 canine adenoviruses.

The recombinant adenoviruses according to the present invention can beprepared using customary techniques which are per se well known to theskilled person (cf., for example, GRAHAM and PREVEC (Manipulation ofAdeno-virus Vectors, Methods Mol. Biol., 7, 109-128, 1991), inparticular using techniques comprising: (i) using standard techniques ofdouble homologous recombination to generate recombinant genomes in E.coli and (ii) transfecting the resulting recombinant genomes intosuitable cell lines which enable said genomes to be amplified andencapsidated in infectious viral particles.

It will be possible, for example, to use homologous recombinationtechniques in E. coli, such as those described by CHARTIER et al., (J.Virol., 70, 7, 4805-4840, 1996) and in U.S. Pat. No. 6110735 in the nameof TRANSGENE or else those described by CROUZET et al., (Proc. Nat.Acad. Sci. USA, 94, 1414-1419, 1997). These methods are based on anintermolecular homologous recombination between a “recipient” DNAmolecule containing the complete genome of an adenovirus and a “donor”DNA molecule comprising a heterologous sequence to be inserted into saidgenome flanked by sequences which are homologous with those of theregion of the adenoviral genome where it is desired to effect theinsertion. The recipient molecule is linearized by cleaving at arestriction site which is unique in the genome of the adenovirus andwhich is located at the insertion site. Selection of the recombinantgenomes is then based on circularization of the recipient molecule.

These methods therefore suffer from the drawback of only being able toinsert said heterologous sequence into a region comprising a restrictionsite which is unique in the genome of the adenovirus.

The inventors have now developed a method for inserting a heterologoussequence into an adenovirus, which method does not require linearizingthe genome of the adenovirus by cleaving at the insertion site.

This method differs from that described by CHARTIER et al. in that:

1) the heterologous DNA fragment (donor molecule) to be inserted intothe genome of the adenovirus (recipient molecule) comprises a selectionmarker which makes it possible to isolate the recombinant plasmids onthe basis of their double resistance to both ampicillin and kanamycin,and

2) said fragment is cotransformed with a recipient molecule which iseither in circular form or in a form which has been linearized bycleaving at a restriction site which is located outside the insertionsite.

Consequently, the present invention also relates to a method forpreparing a recombinant adenovirus by means of intermolecular homologousrecombination in a prokaryotic cell, characterized in that it comprisesthe following steps:

α) introducing, into said prokaryotic cell: (i) a plasmid comprising thegenome of an adenovirus and a first selection gene; and (ii) apreviously linearized DNA fragment which comprises a heterologoussequence to be inserted into said genome flanked by sequences which arehomologous to those flanking the site of said plasmid where theinsertion is to be effected and which includes a second selection genewhich differs from the first; and β) culturing said prokaryotic cellunder selective conditions in order to make it possible to generate andselect cells which harbor recombinant plasmids which are expressing thefirst and second selection genes, and γ) isolating the genome of saidrecombinant adenovirus from selected cells.

“Selective conditions” are understood as meaning culture conditionsunder which the first and second selection agents (for exampleantibiotics) are present at concentrations which do not allowuntransformed cells to multiply but which allow cotransformed cells tomultiply.

According to a first embodiment of the invention, the plasmid which isused in α) is in circular form.

According to a second embodiment of the invention, the plasmid used instep α) has been previously linearized by cleaving at a restriction sitewhich is located outside the insertion site.

According to another advantageous embodiment of the invention, the firstand/or second selection gene is a gene for resistance to an antibiotic,for example a gene for resistance to ampicillin or kanamycin.

According to another embodiment of the invention, the second selectiongene is flanked by 2 identical or different restriction sites which areabsent from the genome of the adenovirus used in step α); this selectiongene can therefore be excised from the sequence of the genome of therecombinant adenovirus by digesting at these sites.

Advantageously, the method according to the invention comprises, afterpreparing the recombinant genome as described in steps α) to γ) above,an additional step of transfecting the recombinant genome into asuitable cell line which enables said genome to be amplified andencapsidated in infectious viral particles.

In order to prepare recombinant adenoviruses according to the invention,it is possible to use cell lines which are known per se to the skilledperson (cf., for example, GRAHAM and PREVEC, see above) and which areexpressing the E1 region of the adenovirus and, where appropriate, theE4 region of the adenovirus when this latter has been impaired byinserting a heterologous sequence of interest. Cell lines which can beused, and which may be mentioned, in particular, are human cell linessuch as the 293 cell line (GRAHAM et al., J. Gen. Virol., 36, 59-74,1977) and canine cell lines such as the DK/E1-28 cell line (KLONJKOWSKIet al., Human Gene Therapy, see above). Advantageously, it will bepossible to use a new cell line which has been constructed by theinventors and which is modified by inserting a fragment consisting ofthe sequence corresponding to that which extends from position 439 toposition 3595 in the genome of the (GenBank J04368) type 2 canineadenovirus; this cell line does not contain the sequences which arelocated upstream of position 439 and which are present in theabove-mentioned cell lines of the prior art.

Said cell line is preferably of canine origin.

The invention additionally relates to plasmids and nucleic acidmolecules which can be used for preparing the genome of a recombinantadenovirus according to the invention, in particular a canineadenovirus, and to said recombinant genomes which an be obtained by themethods as defined above.

The invention relates, in particular, to the following nucleic acidmolecules and plasmids:

-   -   any nucleic acid molecule which is selected from the group        consisting of:        -   a) a nucleic acid molecule which represents the genome of a            recombinant adenovirus according to the invention as defined            above;        -   b) a nucleic acid molecule which consists of a fragment of            the molecule a) above and which comprises between 10 and            1000 bp, preferably at least 300 bp, of the sequence of the            original replicating adenovirus located upstream of the            deleted portion and between 10 and 5000 bp, preferably            between 10 and 1000 bp, preferably at least 300 bp, of the            sequence of the original replicating adenovirus located            downstream of the deleted portion; such a molecule can            additionally comprise all or part of a heterologous sequence            which is inserted in place of the deleted portion or in            proximity thereto.    -   any nucleic acid vector, in particular any plasmid, which        contains an a) or b) nucleic acid molecule as defined above.

The invention also relates to the recombinant adenoviruses according tothe invention for use as drugs.

The invention relates, in particular, to the use of the adenovirusesaccording to the invention for preparing immunogenic or vaccinatorycompositions or drugs which are intended for gene therapy or fortreating cancer as well as for producing recombinant proteins.

Said drug or said composition is preferably intended to be administeredto a wild or domestic carnivore, in particular a cat, dog or fox or elsea human.

The recombinant adenoviruses according to the invention are particularlywell suited for therapeutic uses, for example vaccinatory therapeuticuses, in man and animals. This is because, contrary to thenonreplicating recombinant adenoviruses, whose genome is present at lowcopy numbers per cell and rapidly eliminated from actively dividingcells, the replicating or pseudoreplicating recombinant adenovirusesaccording to the invention multiply significantly in the nucleus of thetransduced cells, making it possible to efficiently transduce bothquiescent cells and actively dividing cells such as tumor cells. Inaddition, the pseudoreplicating adenoviruses according to the invention,which do not produce any infectious particle, exhibit a high degree ofbiosafety and also make it possible to induce a strong immune responsein connection with repeat injections.

Recombinant adenoviruses according to the invention, for example thosederived from the canine adenovirus, have applications for vaccinatingand treating cancer in domestic or wild carnivores, in particular cats,dogs and foxes. In addition, due to a host tropism of their own, thesecanine adenoviruses can be used in human gene therapy for targetingtissues which are different from those which can be transduced by thehuman vectors, for example cells of the central nervous system.

The present invention will be better understood with the help of theremainder of the description which follows and which refers tononlimiting examples which illustrate the construction and preparationof a recombinant adenovirus according to the invention and its use forexpressing genes of interest, in particular for vaccination.

EXAMPLE 1: Constructing the Adenoviruses CAV 311-319, CAV 311-439 andCAV 311-401

1) Constructing the Recombinant Plasmids

The following plasmids were constructed using the standard protocols forpreparing, cloning and analyzing DNA such as those described in CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andSon Inc., Library of Congress, USA)

a) Plasmid pCav2 containing the complete sequence of the genome of type2 canine adenovirus (Cav2)

This plasmid is constructed by homologous recombination in E. coli, inanalogy with the method for preparing human adenoviruses, as describedin CHARTIER et al., see above.

The main steps involved in constructing this plasmid are shown in FIG.1.

More precisely, the left and right ends of the genome of the Manhattanstrain of Cav2, corresponding to the sequences at positions 1 to 1060(fragment A) and 29323-31323(fragment B) are amplified separately by PCRfrom the genomic DNA of the Cav2 Manhattan strain (APPEL et al., Am. J.Vet. Res., 34, 543-550, 1973) using the following primers:

Fragment A 5′-TTGGCGCGCCCATCATCAATAATATACAGGAC- (SEQ ID NO: 1) 3′5′-GCTCTAGACCTGCCCAAACATTTAACC-3′ (SEQ ID NO: 2)

Fragment B 5′-TTGGCGCGCCCATCATCAATAATATACAGGAC- (SEQ ID NO: 1) 3′5′-GCTCTAGAGGGTGATTATTAACAACGTC-3′ (SEQ ID NO: 3)

The resulting fragments A and B are separately cloned into the plasmidpCR2.1 (TA Cloning System, INVITROGEN) in order to give, respectively,the plasmids pCR2.1/left ITR and pCR2.1/right ITR. The plasmidpCR2.1/left ITR is digested with BamHI and XbaI and the 1111 bp fragmentwhich is thus generated is cloned between the BamHI and XbaI sites ofthe plasmid pPolyII Amp^(R) (GenBank M18128, LATHE et al., Gene, 57,193-201, 1987) in order to give the plasmid designated pPolyII/left ITR.The plasmid pCR2.1/right ITR is cleaved with BamHI, treated with Klenowpolymerase and then cleaved with XbaI; the 2052 bp fragment which isthus generated is cloned between the XbaI and PvuII sites of the plasmidpPolyII/left ITR in order to give the plasmid pPolyII.ITRs.Cav2. Thisplasmid contains the left and right ends of the genome of the Cav2Manhattan strain which are cloned in the form of a 3073 bp AscI-AscIfragment comprising an XbaI site in position 1066 of said fragment,making it possible to linearize the plasmid at the DNA insertion site.

The genomic DNA of the Cav2 Manhattan strain and the pPolyII.ITRs.Cav2DNA, which is linearized at the XbaI site, are cotransformed into the E.coli strain BJ5183 recBC sbcBC (HANAHAN et al., J. Mol. Biol., 166,557-580, 1983). A 33425 bp recombinant plasmid, designated pCav2, isisolated from the colonies which are resistant to ampicillin.

Plasmid pCav2 contains the complete genome of Cav2 (Manhattan strain)cloned in the form of a 31331 bp fragment flanked by two AscI siteswhich are unique in this plasmid, with these sites being absent from theCav2 genome (Manhattan and Toronto strains) as well as from that of theovine adenovirus strain OAV.

b) Shuttle plasmids

b₁) pShuttle/311-439/CMVeGFP

This 6111 bp plasmid is derived from the plasmid pBluescript KS(STRATAGENE) by inserting Cav2 sequences upstream and downstream of the312-438 deletion (UpRecSeq 1-311 and DownRecSeq 439-1060) at either endof a cassette for expressing the reporter gene GFP.

This plasmid is constructed in accordance with the following steps:

1) a fragment C, corresponding to the sequence at positions 1 to 311(UpRecSeq) of Cav2, is amplified by PCR using the primers:5′-TTGGCGCCCATCATCAATAATATACAGGAC-3′ (SEQ ID NO: 1)5′-CCGACGTCGACCATAAACTTTGACATTAGCCG- (SEQ ID NO: 4) 3′.The PCR amplification product is cloned into the plasmid pCR2.1 in orderto give the plasmid pCR2.1/UpRecSeq (1-311).

2) a fragment D, corresponding to the sequence at positions 439 to 1060(DownRecSeq) of Cav2, is amplified by PCR using the primers:5′-GCTCTAGAGCGAAGATCTCCAACAGCAATACAC (SEQ ID NO: 5) TCTTG-3′5′-GCTCTAGACCTGCCCAAACATTTAACC-3′. (SEQ ID NO: 2)The PCR amplification product is cloned into the plasmid pCR2.1 in orderto give the plasmid pCR2.1/DownRecSeq (439-1060).

3) A fragment E of approximately 2050 bp, containing the earlycytomegalovirus promoter, an intron, the eGFP (enhanced GreenFluorescent Protein) coding sequence and a polyadenylation signal, isobtained in accordance with the following steps: The plasmid pEGFP-1(CLONTECH) is cleaved with BamHI, treated with Klenow polymerase andthen digested with NotI; the 741 bp fragment which is thus obtained iscloned between the XhoI (previously repaired by treating with the Klenowpolymerase) and NotI sites of the plasmid pCI (PROMEGA) in order to givethe plasmid pCMVeGFP.

The pCMVeGFP plasmid is then cleaved with BglII, treated with Klenowpolymerase and then digested with BamHI in order to generate a 2050 bpfragment (fragment E).

4) The fragment E is inserted between the SmaI and BamHI sites of theplasmid pBLUESCRIPT KS in order to give the plasmid pKS/CMVeGFP. Theplasmid pCR2.1/UpRecSeq (1-311) is cleaved with KpnI and SalI and the371 bp fragment thus obtained (fragment C) is cloned between the KpnIand SalI sites of the plasmid pKS/CMVeGFP in order to give the plasmidpKS/CMVeGFP-C. The plasmid pCR2.1/DownRecSeq (439-1060) is cleaved withXbaI and the 650 bp fragment thus obtained (fragment D) is inserted intothe XbaI site of the plasmid pKS/CMVeGFP-C in order to give the plasmiddesignated pShuttle 311-439/CMVeGFP.

b₂) pShuttle 311-401/CMVeGFP

The shuttle plasmid pShuttle 311-401/CMVeGFP is constructed from theplasmid pShuttle 311-439/CMVeGFP in accordance with the following steps:

The sequence 401-1060 (DownRecSeq) is amplified by PCR using theprimers: 5′-GATAAGGATCACGCGGCCTTAAATTCTCAG-3′ (SEQ ID NO: 6)5′-GCTCTAGACCTGCCCAAACATTTAACC-3′. (SEQ ID NO: 2)The PCR amplification product is cloned in the plasmid pCR2.1 in orderto give the plasmid pCR2.1/DownRecSeq (401-1060).

This plasmid pCR2.1/DownRecSeq (401-1060) is digested with EcoRI andthen treated with Klenow polymerase and the 401-1060 fragment thusobtained is substituted for the 439-1060 fragment of the plasmidpShuttle 311-439/CMVeGFP, which has been previously digested with XbaIand then treated with Klenow polymerase, in order to give the plasmidpshuttle 311-401/CMVeGFP.

b₃) pshuttle 311-319/CMVeGFP

The shuttle plasmid pShuttle 311-319/CMVeGFP is constructed from theplasmid pShuttle 311-439/CMVeGFP in accordance with the following steps:

The 319-1060 sequence (DownRecSeq) is amplified by PCR using theprimers: 5′-GATAAGGATCAACAGAAACACTCTGTTCTCTG- (SEQ ID NO: 7) 3′5′-GCTCTAGACCTGCCCAAACATTTAACC-3′. (SEQ ID NO: 2)The PCR amplification product is cloned into the plasmid pCR2.1 in orderto give the plasmid pCR2.1/DownRecSeq (319-1060).

This plasmid pCR2.1/DownRecSeq (319-1060) is digested with EcoRI andthen treated with Klenow polymerase and the 319-1060 fragment thusobtained is substituted for the 439-1060 fragment of the plasmidpshuttle 311-439/CMVeGFP, which has been previously digested with XbaIand then treated with Klenow polymerase, in order to give the plasmidpshuttle 311-319/CMVeGFP.

b₄) Shuttle plasmid pShuttle 311-439/CMVeGFP/Kana

This 7027 bp plasmid, which is derived from the plasmid pShuttle311-439/CMVeGFP by cloning, into the PmeI site, a cassette forexpressing a gene for resistance to kanamycin in the oppositeorientation to that of the cassette for the GFP, is obtained inaccordance with the following steps:

The reading frame encoding the Kana gene is amplified by PCR from theplasmid pET-29a(+) (NOVAGEN) using the primers:5′-AGCTTTGTTTAAACGGCGCGCCGGGATTTTGGT (SEQ ID NO: 8) CATGAAC-3′5′-CCGGCGCGCCGTTTAAACAAAGCTATCCGCTCA (SEQ ID NO: 9) TGAA-3′.The PCR amplification product is cloned into the plasmid pCR2.1 in orderto give the plasmid pCR2.1-Kana/PmeI. The plasmid pCR2.1/Kana/PmeI iscleaved with EcoRI and treated with Klenow polymerase and the fragmentof approximately 959 bp in size, containing the reading frame encodingthe Kana gene, is inserted into the EcoRV site of the plasmid pshuttle311-439/CMVeGFP in order to give the plasmid pshuttle311-439/CMVeGFP/Kana.

This plasmid pShuttle 311-439/CMVeGFP/Kana, which is depicted in FIG. 2and which contains a gene for resistance to an antibiotic cloned betweenthe adenoviral sequences which are the target of the recombination,upstream of the heterologous sequence to be inserted, advantageouslymakes it possible to select the recombinants which are resistant to bothampicillin and kanamycin. In addition, the Kana gene, which is thenexcised from the recombinant plasmid by digesting at the 2 PmeI sites,is absent from the recombinant adenovirus sequence which is generatedfrom this plasmid.

b₄) Shuttle plasmid pPoly II 311-439/CMVeGFP/Kana (FIG. 2)

This 6332 bp plasmid is obtained by cloning the 4292 bp KpnI-PvuIIfragment of pShuttle 311-439/CMVeGFP between the KpnI (position 42) andPvuII (position 63) sites of the plasmid pPoly II, as illustrated inFIG. 2.

c) Plasmid pCav 311-439/CMVeGFP

This plasmid is obtained by homologous recombination in the E. colistrain BJ5183 in accordance with the following 2 alternatives c1 and c2,which are respectively illustrated by FIGS. 3 and 4:

c₁) Recombination of pShuttle 311-439/CMVeGFP.Kana with plasmid pCav2 incircular form (FIG. 3)

1) the donor molecule, containing the upstream (UpRecSeq 1-311) anddownstream (DownRecSeq 439-1060) recombination sequences and the CMVeGFPand Kana cassettes, is prepared from the plasmid pshuttle311-439/CMVeGFP.Kana by digesting with the restriction enzymes KpnI andEcoRV,

2) the fragment obtained in 1) and the plasmid pCav2 (Amp^(R)) incircular form are cotransformed into the E. coli strain BJ5183, and

3) the recombinant plasmids are isolated on the basis of the criterionof resistance to both ampicillin and kanamycin. The sequence of one ofthem, pCav 311-439/CMVeGFP/Kana, is confirmed by enzymic restriction andby sequencing.

4) The Kana cassette is then excised by digesting with the restrictionenzyme PmeI so as to obtain the plasmid pCav 311-439/CMVeGFP, whichcontains the Cav2 genome from which the 312-438 sequence has beendeleted and replaced with a cassette for expressing GFP.

c₂) Recombination of pPoly II 311-439/CMVeGFP.Kana with the plasmidpCav2, which has been previously linearized outside the insertion site(FIG. 4)

1) the donor molecule is prepared from the ppoly II 311-439/CMVeGFP.Kanaplasmid by digesting with the restriction enzyme SwaI,

2) the plasmid pCav2 is linearized by cleaving at the PvuI site,

3) the fragment obtained in 1) and the linearized pCav2 plasmid obtainedin 2) are cotransformed into the E. coli strain BJ5183, and

4) the recombinant plasmids are isolated on the basis of the criterionof resistance to both ampicillin and kanamycin. The sequence of one ofthem, pcav 311-439/CMVeGFP.Kana, is confirmed by enzymic restriction andby sequencing.

5) The Kana cassette is then excised by digesting with the restrictionenzyme PmeI so as to obtain the plasmid pcav 311-439/CMVeGFP, whichcontains the Cav2 genome from which the sequence at positions 312-438has been deleted, with this sequence being replaced by a cassette forexpressing GFP.

d) Plasmid pCav 311-401/CMVeGFP

The plasmid pshuttle 311-401/CMVeGFP is digested with KpnI and SwaI andthe 3167 bp fragment thus obtained, and the plasmid pcav 311-439 CMVeGFPwhich is linearized at the PmeI site, are cotransformed into the E. colistrain BJ5185. The recombinant plasmid pcav 311-401 CMVeGFP, which isgenerated by homologous recombination, is selected on the basis of thecriterion of resistance to both ampicillin and kanamycin.

e) Plasmid pCav 311-319/CMVeGFP

The plasmid pshuttle 311-319/CMVeGFP is digested with KpnI and SwaI andthe 3249 bp fragment which is thus obtained, and the plasmid pCav311-439 CMVeGFP which has been linearized at the PmeI site, arecotransformed into the E. coli strain BJ5185. The recombinant plasmidpcav 311-319 CMVeGFP, which is generated by homologous recombination, isselected on the basis of the criterion of resistance to both ampicillinand kanamycin.

2) Producing recombinant viruses

The plasmids pcav 311-439/CMVeGFP, pcav 311-401/CMVeGFP or pcav311-319/CMVeGFP are digested with the restriction enzyme AscI in orderto excise the sequences of the recombinant adenovirus genome. Theexcised adenoviral genome is then transformed into the canine cell lineDK/E1-28, which constitutively expresses the Cav2 E1 region (KLONJKOWSKIet al., Human Gene Therapy, see above), in the presence of lipofectamine(GIBCO) in accordance with the customary techniques which are well knownper se to the skilled person (cf., for example, GRAHAM and PREVEC, seeabove). When a cytopathic effect is observed, the virus is harvestedfrom the transfected cells, then amplified in the same DK/E1 -28 cellline and purified by centrifugation through a cesium chloride gradientusing a standard protocol, such as described, for example, in GRAHAM andPREVEC, see above.

The genomic sequence of the viruses Cav 311-439/CMVeGFP, Cav311-401/CMVeGFP and Cav 311-319/CMVeGFP is confirmed by enzymicrestriction and by partial sequencing of the viral DNA, which isextracted from the infected DK/E1 -28 cells and prepared in accordancewith the HIRT technique (J. Mol. Biol., 26, 365-369, 1967). Therecombinant Cav virus preparations are titrated by limiting dilution on96-well plates in accordance with the method of SPEARMAN and KÄRBER(Virology Methods Manual, Brian W J Mahy and Hillar O Kangro, 1996,Academic Press, Harcourt Brace & Company). The TCID ₅₀/ml titer which isobtained by this method is equivalent to the pfu/ml titer which isobtained by the method plaques on DK cells, in accordance with theprotocol described in KLONJKOWSKI et al., see above.

The following results are obtained:

-   -   the isolated viruses Cav 311-439/CMVeGFP, Cav 311-401/CMVeGFP        and Cav 311-319/CMVeGFP have a restriction profile and a        sequence which is in accordance with those which are expected,    -   the purified viruses Cav 311-439/CMVeGFP, Cav 311-401/CMVeGFP        and Cav 311-319/CMVeGFP have a titer of approximately 10^(9.2)        pfu/ml.

EXAMPLE 2: Characterizing the Recombinant Virus Cav 311-439

1) Analyzing the efficiency of transduction and the cytopathic effect ofCav 311-439 CMVeGFP in feline and canine cells

Canine (DK/E1-28 and DK) or feline (CRFK) cell lines are infected withthe virus Cav 311-439 CMVeGFP at a multiplicity of infection of 10pfu/cell. Uninfected cells and cells which are infected with thewild-type Cav virus (Cav2) are used as controls.

At 3 and 5 days after the infection, the presence of a cytopathic effect(EPE) is analyzed by observing infected cells in an optical microscope.In addition, the expression of the transgene by the Cav 311-439 CMVeGFPvirus in the infected cells is confirmed by detecting the GFP byfluorescence microscopy.

The results of this experiment are presented in tables I and II below:TABLE I Virus DK DK/E1-28 CRFK Cav 311-439 CMVeGFP − ++ − Cav + ++ −Control − − −

TABLE II Cells infected with Cav 311-439.CMVeGFP ECP GFP DK − + DK/E1-28++ ++These results show that:

a high level of expression of the transgene by the Cav 311-439 CMVeGFPvirus is observed in the infected cells, and

no cytopathic effect is observed in the unmodified canine cells (DKcells) or feline cells which are infected with this virus Cav 311-439CMVeGFP; a substantial cytopathic effect is only observed in the caninecells which are infected with this Cav 311-439 CMVeGFP virus and whichare expressing the E1 region,

in the controls, a substantial cytopathic effect is observed in thecanine cells (DK and DK/E1-28) which are infected with the wild-type Cav(Cav2) and no cytopathic effect is observed in the feline cells whichare infected with Cav2.

The results of these experiments demonstrate that the Cav 311-439viruses are able to very efficiently transduce the cells withoutinducing any cytopathic effect in the canine cells which are permissivefor replication of the wild-type canine adenoviruses, or in the felinecells.

2) Analyzing the replication of Cav 311-439 CMVeGFP in the feline andcanine cells

Canine (DK/E1 -28 and DK) or feline (CRFK) cell lines are infected withthe virus Cav 311-439 CMVeGFP at a multiplicity of infection of 10pfu/cell. Uninfected cells and cells infected with the wild-type Cavvirus are used as controls.

At 2, 24, 48 and 72 hours after the infection, the cells are harvestedand centrifuged. The intracellular viral DNA is prepared using the HIRTtechnique (J. Mol. Biol., 26, 365-369, 1967), digested with the enzymeEcoRI and then visualized on an agarose gel following electrophoreticmigration.

The results are depicted in FIG. 5:

Legend to FIG. 5: viral DNA extracted from feline (CRFK) or canine (DK,DK/E1-28) cells at different times after infection (2, 24, 48 and 72hours) with the adenovirus Cav 311-439.CMVeGFP is digested with EcoRIand analyzed on an agarose gel. The cell line DK/E1-28, which expressesthe E1 region of the adenovirus, is used as a positive control forreplication.

These results show that:

-   -   the Cav 311-439 CMVeGFP virus replicates its genome in the        tested canine and feline cell lines,    -   the level of replication is greater in the canine cells than in        the feline cells,    -   the peak of replication is reached at 24 hours in the DK/E1-28        cells and at 48 hours in the DK cells, probably because of the        cellular expression of the E1 region in the DK/E1-28 cells.

By comparison, in the control cells infected with the wild-type Cavvirus, a similar quantity of genomic DNA is observed in the 3 celllines.

The results of this experiment demonstrate that the 311-439 deletiondoes not affect the replication of the canine adenovirus: the vectorswhich carry this deletion (Cav 311-439 CMVeGFP) behave like thewild-type adenoviruses as far as the replication of their genome incanine or feline cells is concerned.

3) Analyzing the production of viral particles in the canine cellsinfected with the virus Cav 311-439.CMVeGFP

DK canine cell lines are infected with the vector Cav 311-439 CMVeGFP ata multiplicity of infection of, respectively, 0.1, 1 and 10 pfu/cell.Uninfected cells and cells infected with the wild-type Cav virus areused as controls.

The infected cells are harvested at 2 hours and 6 days after theinfection, and lysed by several cycles of freezing and thawing. The celllysate is titrated by the abovementioned technique of limitingdilutions.

The quantity of virus present in the cells, expressed in pfu/ml, isshown in table III below. TABLE III Virus Time 0.1 pfu/cell 1 pfu/cell10 pfu/cell Cav 311-439 D0 <10^(1.8) 10^(2.4) 10³ D6 <10^(1.8) 10³10^(2.8) Cav D0  10² 10³ 10^(4.4) D6  10^(7.6) 10^(6.8) 10^(6.8)These results show that the Cav 311-439 virus does not produceinfectious viral particles in canine cells, such as the DK cells, whichare not expressing the E1 region: the Cav 311-439 viral cycle isabortive in the canine cells.4) Vaccinating conventional cats with the Cav 311-439 virus

Groups of cats are inoculated intramuscularly with the following dosesof Cav 311-439:

-   -   group 1 (n=2): 9.6 10⁷ pfu    -   group 2 (n=2): 9.6 10⁶ pfu    -   group 3 (n=2): control.

On D14, D21 and D31, the serum anti-eGFP antibodies are titrated byELISA.

The results are depicted in FIG. 6.

Legend to FIG. 6: serum anti-eGFP antibody (Ab) titers in the cat ondays D7, D14, D21 and D31 after the inoculation of different doses ofthe Cav 311-439.CMVeGFP virus: -▪- 9.6 10⁷ pfu/ml (pfu: plaque formingunits), -▴- 9.6 10⁷ pfu/ml, •••□••• 9.6 10⁶ pfu/ml, •••◯••• 9.6 10⁶pfu/ml.

These results show that a single injection of Cav 311-439 induces a(humoral) immune response in the cat which is specific for the gene ofinterest.

EXAMPLE 3: Constructing a Canine-Derived Cell Line Which ConstitutivelyExpresses the Cav2 E1 Region

A new cell line expressing the E1 region is constructed from the DK cellline (immortalized line of dog kidney cells; ATCC CRL 6247) by means ofthe following steps:

The sequence at positions 439 to 3595 of Cav2 (Manhattan strain) isamplified by PCR using the following primers:5′-CGGCCGACTCTTGAGTGCGCAGCGAGA-3′ (SEQ ID NO: 10)5′-GGCGCGCCGAGAGACAACGCTGGACACGG- (SEQ ID NO: 11) 3′.The PCR amplification product is cloned into the plasmid pCR2.1 to givethe plasmid pCR2.1/E1 .

The plasmid pTRE (CLONTECH) is digested with BamHI, treated with Klenowpolymerase and recircularized in order to give the plasmid pTRE/dlBamHI.

The plasmid pCR2.1/E1 is digested with EcoRI and the 3187 bp fragmentwhich is thus obtained is cloned into the EcoRI site of the plasmidpTRE/dl BamHI in order to give the plasmid pTRE E1 Cav2.

This plasmid PTRE E1 Cav2 contains the coding sequence for the E1Aprotein under the control of a minimum CMV promoter and responseelements of the Tet operon (Tet-Responsive Element or TRE), thesequences coding for the E1B proteins (133R and 438R; SHIBATA et al.,Virology, 172, 460-467, 1989) under the control of their own promoterand the endogenous polyadenylation signals for these sequences.

Using pTRE E1 Cav2 as the starting material, a cell line expressing theE1 region is obtained in an analogous manner to that used for obtainingthe DK/E1-28 cell line (KLONJKOWSKI et al., see above).

More precisely, the DK cells are cotransfected with the pTRE E1 Cav2plasmid, which is linearized at the AatII site, and with the plasmidpTK-Hyg (CLONTECH), which is linearized at the AseI site. Clones areselected in the presence of hygromycin (150 μg/ml) and then analyzed bySouthern blotting, Northern blotting, RT-PCR and Western blotting. 4clones which expressed the E1 region (E1A and E1B), and which were ableto efficiently produce the deleted vectors according to the invention,were isolated.

EXAMPLE 4 Immunizing Mice With the Cav 311-319 Virus

Mice which had been divided into three groups were inoculatedintramuscularly with a 108 pfu dose of the following viruses:

-   -   Group 1 (n=4): Cav 311-319 eGFP    -   Group 2 (n=4): Cav 311-319 CONTROL    -   Group 3 (n=1): uninoculated control.

The Cav 311-319 CONTROL virus is isogenic with the Cav 311-319 eGFPvirus apart from the heterologous gene which is inserted (a heterologousgene encoding a protein which does not have any antigenic relationshipwith GFP is inserted in place of the gene encoding GFP).

On D7, the serum anti-eGFP antibodies which were produced by theinoculated mice were titrated by E1 isa.

The results are depicted in FIG. 7.

Legend to FIG. 7: OD at 405 nm for different dilutions (from 1 to 8,respectively, 1/5, 1/15, 1/45, 1/135, 1/405, 1/1215, 1/3645 and 1/10935)of the mouse sera of the following groups: eGFP (♦), CONTROL (▬) andcontrol (▴).

These results show that a single injection of Cav 311-319 induces a(humoral) immune response in the mouse which is specific for theheterologous gene which is inserted in this adenovirus.

1) A recombinant adenovirus which can be obtained from a replicatingadenovirus by deleting all or part of the region of the genome of saidreplicating adenovirus corresponding to that located between positions311 and 499 in the genome of type 2 canine adenovirus (GenBank J04368),with said deletion comprising all or part of the region of the genome ofthe original replicating adenovirus corresponding to that locatedbetween positions 311 and 401 in the genome of type 2 canine adenovirus.2) A recombinant adenovirus as claimed in claim 1, characterized in thatthe deleted portion consists of all or part of the region of the genomeof the original replicating adenovirus corresponding to that locatedbetween positions 311 and 319 in the genome of type 2 canine adenovirus.3) A recombinant adenovirus as claimed in claim 1, characterized in thatthe deleted portion comprises all or part of the region of the genome ofthe original replicating adenovirus corresponding to that locatedbetween positions 318 and 401 in the genome of type 2 canine adenovirus.4) A recombinant adenovirus as claimed in claim 3, characterized in thatthe deleted portion additionally comprises: all or part of the region ofthe genome of the original replicating adenovirus corresponding to thatlocated between positions 311 and 319 in the genome of type 2 canineadenovirus; and/or all or part of the region of the genome of theoriginal replicating adenovirus corresponding to that located betweenpositions 400 and 439 in the genome of type 2 canine adenovirus; and/orall or part of the region of the genome of the original replicatingadenovirus corresponding to that located between positions 438 and 499in the genome of type 2 canine adenovirus. 5) A recombinant adenovirusas claimed in any one of claims 1 to 4, characterized in that itadditionally comprises a heterologous sequence of interest inserted inits genome. 6) A recombinant adenovirus as claimed in claim 5,characterized in that said heterologous sequence is inserted in theregion of the genome corresponding to that located between positions 311and 319 in the genome of type 2 canine adenovirus. 7) A recombinantadenovirus as claimed in any one of claims 1 to 6, characterized in thatit is derived from a type 2 canine adenovirus. 8) A nucleic acidmolecule, characterized in that it is selected from the group consistingof: a) a nucleic acid molecule representing the genome of a recombinantadenovirus as claimed in any one of claims 1 to 7, and b) a nucleic acidmolecule which consists of a fragment of the molecule a) above and whichcomprises between 10 and 1000 bp, preferably at least 300 bp, of thesequence of the original replicating adenovirus located upstream of thedeleted portion and between 10 and 5000 bp, preferably between 10 and1000 bp, preferably at least 300 bp, of the sequence of the originalreplicating adenovirus located downstream of the deleted portion. 9) Aplasmid, characterized in that it comprises a nucleic acid molecule asclaimed in claim
 8. 10) A recombinant adenovirus as claimed in any oneof claims 1 to 7 for use as a drug. 11) The use of a recombinantadenovirus as claimed in any one of claims 1 to 7 for preparing a drugwhich is intended for gene therapy. 12) The use of a recombinantadenovirus as claimed in any one of claims 1 to 7 for preparing a drugwhich is intended for treating cancer. 13) The use of a recombinantadenovirus as claimed in any one of claims 1 to 7 for preparing animmunogenic or vaccinatory composition. 14) The use as claimed in anyone of claims 11 to 13, characterized in that said drug or saidcomposition is intended to be administered to a domestic or wildcarnivore. 15) The use as claimed in any one of claims 11 to 13,characterized in that said drug or said composition is intended to beadministered to humans. 16) A method for preparing a recombinantadenovirus by means of intermolecular homologous recombination in aprokaryotic cell, characterized in that it comprises the followingsteps: α) introducing, into said prokaryotic cell: (i) a plasmidcomprising the genome of an adenovirus and a first selection gene; and(ii) a previously linearized DNA fragment which comprises a heterologoussequence flanked by sequences which are homologous to those flanking thesite of said plasmid where the insertion is to be effected and whichincludes a second selection gene which differs from the first; and β)culturing said prokaryotic cell under selective conditions in order tomake it possible to generate and select cells which harbor recombinantplasmids which are expressing the first and second selection genes, andγ) isolating the genome of said recombinant adenovirus from selectedprokaryotic cells. 17) The method as claimed in claim 16, characterizedin that the plasmid employed in step α) is in circular form. 18) Themethod as claimed in claim 16, characterized in that the plasmidemployed in step α) has been previously linearized by being cleaved at arestriction site which is located outside the insertion site. 19) Themethod as claimed in any one of claims 16 to 18, characterized in thatsaid second selection gene is flanked by 2 identical or differentrestriction sites which are absent from the genome of the adenoviruswhich is included in the plasmid employed in step α). 20) The method asclaimed in any one of claims 16 to 19, characterized in that itcomprises an additional step of transfecting said recombinant genomeinto a cell line which enables said genome to be amplified andencapsidated in infectious viral particles. 21) The use of a recombinantadenovirus as claimed in any one of claims 1 to 7 for producingrecombinant proteins.